1. Field of the Invention
This invention in the fields of biochemistry and medicine is concerned with chemically crosslinked Staphylococcal protein A, Staphylococcal enterotoxins or functional derivatives thereof and their use alone or in combination with immunoglobulins or complement components in the treatment of autoimmune and neoplastic diseases.
2. Description of the Background Art
Protein A is a constituent of the cell wall of many strains of bacteria of the species Staphylococcus aureus. This protein (abbreviated xe2x80x9cSpAxe2x80x9d herein) has molecular weight of 42 kDa and binds selectively to immunoglobulins (Igs), particularly IgG, and immune complexes from many mammalian species. Ig binding sites for SpA are located in the Fc region of the Ig molecule. SpA-Ig complexes display diverse biological activities including complement binding and activation (Langone, J. J., Adv. Immunol. 32:157-252 (1982)).
An immunoadsorbent column consisting of SpA immobilized on collodion-coated charcoal was originally used for ex vivo immunoadsorption and provided successful extracorporeal treatment for patients with breast carcinoma (Terman, D. S., U.S.Pat. No. 5,091,091, Feb. 2, 1995). Subsequently, a SpA-silica immunoadsorbent column was developed by Bensinger, Kinet and others (Bensinger, U.S. Pat. No. 4,614,513., Sep. 30, 1986; Bensinger W. I. et al., New Eng. J Med 306:935 (1982); Kinet, J. P. et al., Eur. J Clin Invest. 16:50-55 (1986)) and later by Balint et al.(U.S. Pat. No. 4,681,870, Jul. 21, 1987). This column, known by its trade name of Prosorba(copyright), produced by IMRE Corporation, has received FDA approval for treatment of idiopathic thrombocytopenic purpura (ITP) and hemolytic-uremic syndrome. Prosorba(copyright) columns have also been reported to show efficacy against advanced cancer and autoimmune diseases such as rheumatoid arthritis (RA) (Balint et al., supra; Snyder, H. W. et al., J Clin. Apheresis 7:110:118 (1992); Snyder, H. W. et al., Sem. Hematol 26:31 (1989); Mittelman, A. et al., Sem. Hematol. 26:15 (1989); Messerschmidt, G. L. et al., Sem. Hematol. 26:19 (1989); Snyder, H. W. et al, J Clin. Apheresis 6:1 (1991); Snyder, H. W. et al., Blood 79:2237 (1992)). Clinically, these columns may be used in an xe2x80x9con-linexe2x80x9d or xe2x80x9coff-linexe2x80x9d mode with identical therapeutic effects. In the off-line mode, 200 ml of plasma is collected by phlebotomy from a subject, passed over the column and then returned to the same donor. Publications sponsored by the manufacturer of the column (IMRE Corporation) indicate that the coupling of SpA to silica creates a xe2x80x9cstable covalent bondxe2x80x9d such that the bound SpA is not released into the perfused plasma (Snyder, H. W. et al., J Clin. Apheresis 7:110:118 (1992); Snyder, H. W. et al., Sem. HematoL 26:31 (1989); Mittelman, A. et al., Sem. Hematol. 26:15 (1989); Messerschmidt, G. L. et al, Sem. Hematol. 26:19(1989); Snyder, H. W. et al., J Clin. Apheresis 6:1 (1991). Snyder, H. W. et al., Blood 79:2237 (1992); Balint, J. P., Blood 84:664 (1994)). The removal of immune complexes from plasma perfused over the column is said to be the basis of the column""s therapeutic effect. However, despite the purported covalent bonding of the SpA to the column matrix, several investigators (including the manufacturer) have reported that SpA does indeed leach from the column surface into the perfused plasma in amounts that have been reported to vary from 200 xcexcg to 1 mg of SpA per treatment dose of perfused plasma (Sato, H. et al., Transfusion Sci. 12:299 (1991); Kinet, J. P. et al., Eur. J Clin. Invest. 16:43 (1986); IMRE Corporation FDA Safety and Efficacy Report on Prosorba(copyright) Column, 1987).
The clinical toxicity of the procedure (Smith, E. et al., J Clin. Apheresis 7:4 (1992); Ciavarella, D. et al., Int. J Clin. Lab. Res. 21:210 (1992); Dzic, W., New Eng. J Med 331:792 (1994)) and the known in vivo and in vitro biological effects of SpA (Langone, J. J., J Biol. Resp. Modif. 3:241 (1984)) might have hinted that leached SpA was of biotherapeutic significance. Nevertheless, according to the present state of the art, there is no pharmacologic significance ascribed to the leached material. Rather, the art teaches that the therapeutic effects of these columns are due entirely to the adsorption and removal by the column of immune complexes from plasma (supra). Accordingly, there has been no attempt, prior to the work leading up to the present invention, to examine carefully the composition of products leaching from these columns or to characterize their molecular structure and biologic activity.
IMRE Corporation scientists have demonstrated antitumor effects in human cancer patients (overall response rate of 30%) using the Prosorba(copyright) column. The best responses were seen in patients with breast cancer and Kaposi""s sarcoma (Messerschmidt, G. L. et al., J Clin. Oncol. 6:203-212 (1988)). The authors ascribed the effect to adsorption of circulating immune complexes from tumor bearing plasma to the Prosorba(copyright) column. However, as viewed by the present inventor in light of the invention disclosed herein, it is more likely that the effects were due to complexes of (1) leached SpA and IgG, (2) leached SpA and Staphylococcal enterotoxin B (SEB) and/or (3) SpA, SEB and IgG. The variability of patient responses is likely due to the unpredictability and the broad range of the amount of leached polymeric or complexed SpA emerging from the column.
Enterotoxins are known to produce T cell anergy or sensitization depending on the dose (Sundstedt, A. et al., J Immunol. 154:6306-6313 (1995)). For example, very large or very small doses of enterotoxins produce anergy whereas intermediate doses produce sensitization. As noted above, the effectiveness of Prosorba(copyright) in the treatment of an autoimmune disease (ITP, RA) and cancer was thought to be due to the adsorption and removal of immune complexes by the column.
Based on the present invention, the contention that the therapeutic effects of Prosorba(copyright) (in ITP, for example) are due to xe2x80x9cimmune complex removalxe2x80x9d from 200 ml of plasma by the column would seem far-fetched. According to such reasoning, the mere removal of this volume of plasma by phlebotomy (the first step of Prosorba(copyright) treatment) should show the same or greater therapeutic efficacy since withdrawing 200 ml of blood effectively, by definition, removes 100% of the immune complexes contained therein. However, neither phlebotomy alone nor plasmapheresis effects any change in the clinical course of ITP or other diseases in which Prosorba(copyright) has been effective. A comparative study of the therapeutic effects of the two treatments in ITP further corroborates this assertion (Kiprov, D. D. et al., J Clin. Apheresis 3:133 (1986)).
The present inventor is the first to have found that the basis for therapeutic activity of the Prosorba(copyright) column in ITP and RA is not what it is claimed to be. Rather, by analyzing what elutes from these columns under various conditions, including conditions of standard clinical use, the present inventor has discovered novel compositions which represent significant, totally unexpected improvements in the treatment of autoimmune diseases such as ITP and RA and of cancer. In making this invention, the present inventor has characterized this eluted material and has designed novel therapeutic compositions and methods for treating autoimmune and neoplastic diseases.
The present inventor has identified SpA in perfusates of SpA immunoadsorbent columns and has shown that it originates from material associated with the column which is not covalently bound to the column matrix. The fundamental discovery for the present invention, was that plasma emerging from the SpA column contained SpA that could be in a monomeric form or in a polymerized, crosslinked high molecular weight form. In fact, SpA which desorbs from the column using various forms of mild perfusion is predominantly in the form of SpA oligomers or polymers. These discoveries were made using a SpA immunoadsorbent column prepared in the laboratory under conditions similar to those specified by the IMRE Corporation and approved by the FDA for clinical use.
When administered to patients with autoimmune disease or cancer, such leached monomeric SpA or SpA polymers readily combine with IgG in host plasma to form high molecular weight SpA-IgG complexes. The therapeutic effects of the SpA polymers of the present invention in ITP patients are believed to be due to the prior complexing of the SpA polymers with IgG. These complexes, mimicking immune complexes, bind to FcR of macrophages, lymphocytes and platelets (Dima, S. et al., Eur. J. Immunol. 13:605 (1983); Kinet, J. P. et al., In: Human Neoplasms, in Selective Plasma Component Removal, A. Pineda, ed., pp. 105 (1984); Sulica, A. et al., Immunology 38:173 (1979); Hawiger, J. et al., J Clin. Invest. 64:931 (1979); Kay, H. S. et al., J Immunol. 118:2058 (1977); Dosset, J. H. et al., J Immunol. 103:1405 (1969); Austin, R. M. et al., J Immunol. 117:602 (1976); Forgsgren, A. et al., J Immunol. 112:1177 (1974)). Indeed, complexes made up of polymeric SpA and IgG are far more effective than previously described monomeric SpA-IgG complexes in blocking FcR-associated functions (Terman, D. S. et al., U.S. Pat. No. 4,699,783, Oct. 13, 1987). Hence, these polymers are extremely efficient in FcR blocking and can achieve the pharmacologic effects at relatively low doses, thereby minimizing the systemic toxicity associated with administration of larger amounts of SpA (which may be due to the SpA itself or to contaminating molecules such as enterotoxins).
Based on the observations of the present inventor disclosed herein, the clinical effects of Prosorba(copyright) may be related in part to the amount of SEB complexed with and accompanying the leached SpA polymers. Ratios of SpA to enterotoxin B of about 1000 (by weight) will produce antitumor effects. In contrast, ratios  greater than 1000, with SEB in quantities  greater than 1 ng/ml of column perfusate, by inactivating T cells may not result in antitumor effects. Leached SEB in intermediate amounts, between 1 ng/ml and 1 xcexcg/ml may result in T cell desensitization and anergy.
Indeed, according to the present invention, the therapeutic effect of Prosorba(copyright) in autoimmune diseases is due in part to the induction of (1) T cell anergy by leached enterotoxins and (2) B cell tolerance by high molecular weight crosslinked SpA polymers. The effects of Prosorba(copyright) in cancer is due to systemic delivery of sensitizing doses of enterotoxins present in the polymerized SpA complexes. B cells are known to express Fcxcex3 receptors with affinity for SpA. Although monomeric SpA is a potent B cell mitogen, high polymerized covalently crosslinked SpA is thought to induce B cell anergy, even in small quantities.
According to the present invention, organic chemical methods, preferably carbodimide crosslinking are used to produce crosslinked SpA molecules. These chemically crosslinked xe2x80x9coligomers or polymersxe2x80x9d are then employed therapeutically, preferably (a) by direct intravenous injection or (b) by prior complexing with IgG, with specific antibodies (preferably the IgG isotype) or with complement components followed by administration to a subject. Hence, this invention permits the achievement of the positive clinical effects of SpA column xe2x80x9cimmunoadsorptionxe2x80x9d procedures without the need for the expensive columns, the manpower and technical expertise required to carry out the column preparation and perfusion procedures, the unpredictable quantity of leached products, and the toxic side effects accompanying the column-based therapy. By prescribing the amount and form of SpA to be infused to a patient, the present invention avoids the toxicity while improving the therapeutic index of the agent.
Specifically, the present invention is directed to a therapeutic composition useful for treating an autoimmune or neoplastic disease comprising a mixture of monomeric and crosslinked polymeric SpA molecules or a functional derivative of a SpA molecule, wherein
(a) the cross-linked polymer molecule comprises at least two monomeric units of SpA or of the functional derivative; and
(b) at least 10% of the total protein or functional derivative is in the form of polymers In the above composition, the crosslinked SpA or functional derivative molecules preferably comprise polymers having a range of molecular masses from about 12 kDa to about 10,000 kDa.
In another embodiment of the above composition, the average molecular mass of the polymers is at least 500 kDa and at least about 50% of the total SpA or functional derivative is in the form of polymers. In yet another embodiment, the average molecular mass of the polymers is between about 64 kDa and about 1000 kDa; and at least about 70% of the total SpA or functional derivative is in the form of polymers. Alternatively, the average molecular mass of the polymers is between about 64 kDa and about 10,000 kDa; and at least about 90% of the total SpA or functional derivative is in the form of polymers.
The present invention also provides the above composition wherein the polymeric SpA or functional derivative is complexed with immunoglobulin (Ig) molecules to form a polymeric SpA-Ig complex. Other useful a therapeutic compositions comprise the polymeric SpA-Ig complexes further complexed with complement components to form a SpA-Ig-complement complex. The Ig is preferably IgG. The IgG may be a purified specific antibody, including a mAb.
The present invention is directed to a polymeric SpA composition wherein the SpA or functional derivative is further crosslinked with molecules of a bacterial superantigen or a functional derivative thereof to form a mixture of
(a) polymeric crosslinked SpA or SpA functional derivative,
(b) polymeric crosslinked conjugates of
i. SpA-superantigen,
ii. SpA functional derivative-superantigen,
iii. SpA-superantigen functional derivative, or
iv. SpA functional derivative-superantigen functional derivative; and
(c) polymeric crosslinked superantigen or superantigen functional derivative. The bacterial superantigen is preferably selected from a group consisting of an enterotoxin of Staphylococcus aureus, toxic shock syndrome toxin, a Streptococcus pyrogenic exotoxin, a Mycoplasma arthritides toxin and a Yersinia enterocolitica toxin.
Also provided is a composition useful for treating an autoimmune or neoplastic disease comprising a chemically crosslinked polymer of SpA, or of a functional derivative of SpA, and having the following characteristics: (a) immunoglobulin Fc binding activity is less than half that of native SpA; and (b) immunoglobulin VH3 region binding is more than about twice that of native SpA. The SpA polymer may be further crosslinked to molecules of a bacterial superantigen or functional derivative thereof The present invention is further directed to a method for preparing a therapeutic compositions as described above, which comprises treating SpA or the functional derivative with a crosslinking agent under conditions which result in crosslinking of the SpA or the functional derivative or both to produce the composition.
A preferred embodiment of the method comprises treating a mixture of SpA and a bacterial superantigen with a crosslinking agent under conditions which results in crosslinking of any one of (a) SpA or its functional derivative with like molecules; (b) SpA with superantigen; (c) SpA functional derivative with superantigen; (d) SpA with superantigen functional derivative; (e) SpA functional derivative with superantigen functional derivative; or (f) superantigen or its functional derivative with like molecules.
Another embodiment provides a method for reducing the toxicity of monomeric enterotoxin molecules, comprising mixing the enterotoxin molecules with SpA or an SpA functional derivative, adding a crosslinking agent capable of crosslinking the enterotoxin molecules and the SpA and allowing any enterotoxin to be chemically crosslinked with the SpA, thereby reducing the toxicity. Alternatively, toxicity can reduced by simply crosslinking enterotoxin molecules with themselves.
In the foregoing methods, the crosslinking agent is selected from the group consisting of a carbodiimide, a homobifunctional aldehyde, a homobifunctional epoxide, a homobifunctional imidoester, a homobifunctional N-hydroxysuccinimide ester, a homobifunctional maleimide, a homobifunctional alkyl halide, a homobifunctional pyridyl disulfide, a homobifunctional aryl halide, a homobifunctional hydrazide, a homobifunctional diazonium derivative and a homobifunctional photoreactive compound.
The above crosslinking agent is preferably a carbodiimide selected from the group consisting of 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide, (1-ethyl-3-(3-dimethyaminopropyl carbodiimide (EDC) and 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.
The crosslinking agent may also be a heterobifunctional compound selected from the group consisting of compounds having:
(a) an amine-reactive and a sulfhydryl-reactive group;
(b) an amine-reactive and a photoreactive group; and
(c) a carbonyl-reactive and a sulfhydryl-reactive group.
In the crosslinking method may be performed by coupling a carbonyl group to an amine group or to a hydrazide group by reductive amination.
Also provided is a method for preparing a therapeutic composition comprising cross-linking the mixture of SpA or the functional derivative and the superantigen, as described above, with a carrier, which carrier consists of a protein, lipid or other polymer which can be covalently bonded to the SpA or the derivative and the superantigen, thereby creating a heterogeneous polymer complex. When the carrier is a protein it is preferably serum albumin, keyhole limpet hemocyanin, tetanus toxoid, ovalbumin, thyroglobulin, diphtheria toxoid, myoglobin, immunoglobulin or purified protein derivative of tuberculin. The carrier may be polymer selected from the group consisting of a polysaccharide, a poly(amino acid), a poly(vinylalcohol), a polyvinylpyrrolidone, a poly(acrylic acid), a polyurethane and a polyphosphazene.
In one embodiment of the above method, the crosslinking produces a SpA polymer or SpA-superantigen polymer covalently bonded to a liposome.
In the foregoing methods, the bacterial superantigen is preferably an enterotoxin of Staphylococcus aureus, toxic shock syndrome toxin, a Streptococcus pyrogenic exotoxin, a Mycoplasma arthritides toxin or a Yersinia enterocolitica toxin.
In the above method for preparing a therapeutic composition comprising a complex between polymeric SpA or a functional derivative thereof and Ig, the method preferably comprises incubating a composition as described above with a fluid containing Ig and allowing the formation of the complex. The fluid is preferably plasma and the Ig is preferably IgG. In one embodiment, the fluid preferably contains a specific antibody of the IgG isotype.
The present invention provides a method for preparing a therapeutic composition useful to for treating an autoimmune or neoplastic disease which composition comprises a complex between polymeric SpA or a functional derivative thereof, Ig and complement, the method comprising
(a) incubating a mixture of monomeric and crosslinked polymeric SpA molecules or a functional derivative of a SpA molecule, as above, with a fluid containing Ig, preferably IgG, to produce a mixture containing Ig bound to the polymeric SpA or functional derivative;
(b) adding to the mixture of step (a) complement or components thereof and allowing the formation of the complex.
The invention is also directed to a pharmaceutical composition useful for treating a subject with an autoimmune or neoplastic disease, comprising
(a) an effective amount of a composition as described above; and
(b) a pharmaceutically acceptable excipient or carrier. The composition may further be bonded to or sequestered within a liposome vesicle.
Also provided herein is a method of treating a subject with an autoimmune disease or cancer, comprising administering to the subject the above pharmaceutical composition.
In a method of extracorporeal treatment of a subject having an autoimmune or neoplastic disease, wherein, over a course of one or more treatments, plasma of the subject is perfused through a SpA-silica immunoadsorbent column to produce a perfusate, and the perfusate is reinfused to the subject, the present invention provides an improvement which comprises:
(a) over the course of one or more treatments, perfusing plasma of the subject through the column;
(b) at each treatment, measuring the amount of SpA in the perfusate prior to the reinfusing;
(c) when the amount of total SpA in the perfusate is less than about 2 xcexcg, increasing the volume of the perfusate or increasing the number of doses of the perfusate such that the subject receives between about 2 xcexcg and about 200 xcexcg per treatment;
(d) when the amount of total SpA in the perfusate is greater than about 200 xcexcg, decreasing the volume of the perfusate such that the subject receives between about 2 xcexcg and about 200 xcexcg per treatment; thereby treating the subject. In another embodiment of the above extracorporeal treatment method, the improvement comprises:
(a) over the course of one or more treatments, perfusing plasma of the subject through the column;
(b) at each treatment, measuring the amount of bacterial enterotoxins in the perfusate prior to reinfusing;
(c) when the amount of total bacterial enterotoxins in the perfusate is less than about 1 ng, increasing the volume of the perfusate or increasing the number of doses of the perfusate such that said subject receives between about 1 ng and about 200 ng per treatment;
(d) when the amount of total bacterial enterotoxins in the perfusate is greater than about 200 ng, decreasing the volume of said perfusate such that the subject receives between about 1 ng and about 200 ng per treatment, thereby treating the subject.